The present invention relates to antisense oligonucleotides complementary to mRNA of urokinase human receptor and to pharmaceutical compositions containing them as the active ingredients.
Plasminogen activation is considered a central process in the regulation of pericellular proteolysis which occurs under both normal and pathological conditions, including cancer invasion, when tissue destruction and cell migration are required. Even though the roles of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) in in vivo neoplasias and in vitro transformed cells have not yet been elucidated, however, it is now well established that u-PA plays a paramount role in neoplastic cell transformation (Physiol. rev. 73(1): 161-195, 1993).
After secretion as a zymogen (pro-u-PA), the active form is obtained by cleavage at lysine 158 into two chains connected by a disulfide bridge. As a result of their interaction with a specific receptor (u-PAR) (Advances in Cancer Res. 57:273-328, 1991) which belongs to the glycosyl-phosphatidyl inositol-anchored proteins family (Exp. Cell. Res. 203:427-434, 1992), pro-u-PA and the active enzyme equilibrate between solution and the cell surface. (Pro)-u-PA binds to the receptor by a sequence framed within the amino-terminal fragment of the A chain thus exposing the B chain, which contains the catalytic site, to the extracellular milieu. On some cell lines, growing as monolayers in vitro, urokinase receptors are located at specific sites on the ventral side of the cell: the so-called focal contacts. Other components of the plasminogen activation system have been identified on the extra-cellular side of cell contacts and have been suggested to play a regulatory role in the modulation of cell-substratum adhesion and extra-cellular matrix destruction. In particular, the presence of u-PA at the main contact sites of the cell has been considered critical in determining the detachment of the cell from the substratum and progression within tissues. Using cell monolayers, it has been observed that the adhesion sites specifically concentrate u-Pa binding sites (Advances in Cancer Res. 57:273-328, 1991).
A number of evidences exist that the expression of urokinase receptor is a basical factor of the invasive characteristics of tumour cells. It has particularly been suggested that the contribution of u-PAR to the invasive phenotype may involve the increased plasmin production by receptor-bound u-PA (J. Biol. Chem. 266: 752-758, 1991). Some authors (Cancer Res. 53:3139-3117, 1993) reported that the overexpression of an exogenous cDNA coding for the u-PA binding site in a human osteosarcoma cell line induced the increase of cell migration in the matrigel test, in addition to increased laminin degradation. In another study (Proc. Natle. Acad, Sci. USA 90:5021-5025, 1993), it was shown that displacement of receptor bound u-PA by a mutant u-PA lacking enzymatic activity, caused the reduction of brain, lung and regional lymph nodes metastasis of prostate cancer cells PC-3. Still other authors (Br. J. Cancer 67:537-544, 1993) have shown that the matrigel invasion by the ovarian cancer cell lines HOC-1 was inhibited by peptides corresponding to the u-PA receptor binding fragment.
It is therefore evident that the u-PA/u-PAR system may be a target for drugs which, decreasing or blocking the invasive action of the cells, may be useful as anti-tumor and anti-metastatic agents.
It has been already shown, for instance, that antibodies against human u-PA prevent the metastasis formation in chicken embryonal lung of human carcinoma cells (Cell 35:611-619, 1983). The anti-messenger strategy has also been used against the u-PA gene in NIH 3T3 cells: in such a case, the selective block of the u-PA gene induced in the cells an alternative invasive mechanism based on the gene expression of another protease, cathepsin L (Cancer Res. 52:6682-6689, 1992).